Chlamydial vaccines and immunogenic compositions containing an outer membrane antigen and methods of preparation thereof

ABSTRACT

Immunogenic compositions including vaccines are described that comprise an outer membrane antigen extract of a strain of Chlamydia and are effective in protection against disease caused by Chlamydia infection The immunogenic compositions may comprise the major outer membrane protein (MOMP) of Chlamydia which may be in a homooligomeric form or complexed with at least one other antigen of Chlamydia. The immunogenic composition may include an immunostimulating complex (ISCOM) and the outer membrane antigen may be incorporated therein. The immunogenic compositions have utility as chlamydial vaccines and in diagnostic applications.

REFERENCE TO RELATED APPLICATION

This application is a U.S. national phase application under 35 U.S.C.371 of PCT/CA97/00656 filed Sep. 11, 1997 which is acontinuation-in-part of U.S. patent application No. 08/713,236 filedSep. 12, 1996.

FIELD OF INVENTION

The invention relates to the field of immunology and, in particular,relates to vaccines against Chlamydia.

BACKGROUND TO THE INVENTION

Chlamydia trachomatis is a species of the genus Chlamydiaceae, orderChlamydiales. C. trachomatis infects the epithelia of the conjunctivaeand the genital tract, causing trachoma and a variety of sexuallytransmitted diseases (STDs) which can lead to, respectively, blindnessor infertility. There are at least 15 serovars of C. trachomatis, ofwhich A, B, and C are causative agents of trachoma, while serovars D, E,F, G, H, I, J, and K are the most common causative agents of chlamydialSTDs. C. trachomatis infections are endemic throughout the world.Trachoma is the leading cause of preventable blindness in developingnations, and it is estimated that 600 million people suffer fromtrachoma worldwide, with as many as 10 million of them being blinded bythe disease. In the United States there are an estimated 3 million casesper year of STDs caused by C. trachomatis.

The pathogenesis of trachoma involves repeated ocular infections and thegeneration of a deleterious hypersensitivity response to chlamydialantigen(s) (refs 1 to 4—Throughout this specification, variousreferences are referred to in parenthesis to more fully describe thestate of the art to which this invention pertains. Full bibliographicinformation for each citation is found at the end of the specification,immediately following the claims. The disclosures of these referencesare hereby incorporated by reference into the present disclosure). Theavailable evidence supports the hypothesis that both secretory IgA andcell-mediated immune responses are important components of protection.Ocular infection in a primate model induces rapid and persistentproduction of IgA in tears, whereas the presence of IgG in tears istransient, corresponding to the period of peak conjunctival inflammation(ref. 5). Protective immunity following experimental ocular infection ina sub-human primate model is homotypic and resistance to ocularchallenge correlates with the presence of serovar-specific antibodies intears (refs. 1, 2, 6). Tears from infected humans neutralised theinfectivity of homologous but not heterologous trachoma serovars for owlmonkey eyes (ref. 7) whereas passive humoral immunization withantitrachoma antibodies was not protective (ref. 8). Several lines ofevidence indicate the importance of cell-mediated responses inprotection from or clearance of chlamydial infection. B-cell deficientmice can resolve infection, whereas nude mice become persistentlyinfected. Adoptive transfer of at least some chlamydia-specific T-celllines or clones can cure persistently infected nude mice, and thisanti-chlamydial activity is probably a function of the ability of theT-cells to secrete interferon-γ(refs. 9 to 17).

Past attempts to develop whole-cell vaccines against trachoma haveactually potentiated disease by sensitizing vaccinees (refs. 1, 2).Sensitization has been determined to be elicited by a 57 kD stressresponse protein (SRP) (HSP60) present in all serovars of C.trachomatis. Repeated exposure to the 57 kD SRP can result in a delayedhypersensitivity reaction, causing the chronic inflammation commonlyassociated with chlamydial infections. Thus, an immunogenic preparationcapable of inducing a strong and enduring mucosal neutralising antibodyresponse and a strong cellular immune response without sensitizing thevaccinee would be useful (ref. 18).

A most promising candidate antigen for the development of a vaccine isthe chlamydial major outer membrane protein (MOMP) (refs. 19 to 21).Other surface proteins and the surface lipopolysaccharide are alsoimmunogenic, but the antibodies they induce have not been found to beprotective (ref. 22, 23). The MOMP, which is the predominant surfaceprotein, is an integral membrane protein with a mass of about 40 kDawhich, with the exception of four variable domains (VDs) designated I,II, III, and IV, is highly conserved amongst serovars. The sequences ofall four VDs have been determined for fifteen serovars (ref. 24, 25).Antibodies capable of neutralising chlamydial infectivity recognize theMOMP (ref. 26, 27, 28, 29). Epitopes to which MOMP-specific neutralisingmonoclonal antibodies bind have been mapped for several serovars (refs.22, 23, 30, 31, 32, 33, 34), and represent important targets for thedevelopment of synthetic or subunit vaccines. The binding sites arecontiguous sequences of six to eight amino acids located within VDs I orII, and IV, depending on the serovar. Subunit immunogens (e.g. isolatedMOMP or synthetic peptides) containing MOMP epitopes can induceantibodies capable of recognising intact chlamydiae (ref. 26). However,conventionally administered subunit immunogens are generally poorinducers of mucosal immunity. It would be useful to formulate chlamydialantigens in such a way as to enhance their immunogenicity and to elicitboth humoral and cell-mediated immune responses.

Immune stimulating complexes (ISCOMs) are cage-like structures formedfrom a mixture of saponins (or saponin derivatives), cholesterol andunsaturated fatty acids. The components of ISCOMs are held together byhydrophobic interactions, and consequently proteins which are naturallyhydrophobic (such as MOMP) or which have been treated to expose or addhydrophobic residues can be efficiently incorporated into the ISCOMs asthey form (ref. 35, 36, 37).

C. trachomatis naturally infects the mucosal surfaces of the eye andgenital tract, and secretory IgA cellular responses are probablyimportant components of protection. Consequently, it would be useful fora chlamydial vaccine to induce a mucosal immune response including bothcellular and antibody components.

C. trachomatis infection may lead to serious disease. It would beadvantageous to provide outer membrane antigen extracts of Chlamydia,including the major outer membrane protein of Chlamydia, particularly insubstantially the native conformation for antigens in immunogenicpreparations including vaccines, and immunogens and the generation ofdiagnostic reagents.

SUMMARY OF THE INVENTION

The present invention provides a novel immunogenic form of chlamydialMOMP which is useful in providing protection against chlamydialdiseases, as well as methods of preparing such materials.

In accordance with one aspect of the invention, there is provided animmunogenic composition, comprising an outer membrane antigen extract(MAE) of a strain of Chlamydia, which may be Chlamydia trachomatis, andan immunostimulating complex (ISCOM).

The MAE may comprise the major outer membrane protein (MOMP) of thestrain of Chlamydia. The MOMP may be in an oligomeric form and/or may becomplexed with at least one other antigen of the strain of Chlamydia.Such oligomers and complexes may have a molecular weight of from about45 to about 125 kDa. The procedure described herein for preparation ofthe MAE, specifically MOMP, produces material which is substantiallyfree from the heat shock protein HSP60 of the strain of Chlamydia. Theimmunogenic composition provided herein may be in the form of the MEAincorporated into ISCOMs.

The immunogenic compositions provided herein may be employed, inaccordance with another aspect of the invention, to protect a hostagainst disease caused by a strain of Chlamydia by administering to thehost an effective amount of the immunogenic composition. Suchadministration may be to a mucosal surface to produce a mucosal immuneresponse. Alternatively, any other convenient means of administrationmay be employed to produce the desired immune response. Theadministration may be to the mucosal surface of the host by intranasaladministration and may produce a genital tract immune response. Inaddition, the immunogenic composition provided herein may be employed asa booster immunization in a prime-boost immunization procedure in whichthe prime immunization is effected with same form of Chlamydia, such asattenuated strain or a vector, including viral and bacterial vectors,containing a chlaymdial gene-expressing a chlamydial protein.

The present invention further includes a method of producing an outermembrane antigen extract of a strain of Chlamydia using a combination oftwo or more detergents including non-ionic detergents. Accordingly, in afurther aspect of the invention, there is provided such a method whichcomprises:

detergent extracting elementary bodies (EBs) of the strain of Chlamydiain the presence of a reducing agent for disulphide bonds to solubilizecytoplasmic material away from outer membrane material;

separating the solubilized cytoplasmic material from the outer membranematerials,

detergent extracting the outer membrane material using at least twonon-ionic detergents in the presence of a reducing agent for disulphidebonds to solubilize outer membrane antigens; and

separating the solubilized outer membrane antigens from residualunextracted membrane-associated material to provide the outer membraneantigen extract.

In one embodiment of this aspect of the invention, the at least twonon-ionic detergents comprise a N-methyl glucamide non-ionic detergentwhich may be selected from heptanoyl-, octanoyl-, nonanoyl- anddecanoyl-N-methyl glucamide, and a glucopyranoside non-ionic detergent,which may be selected from n-hexyl-β-D-, n-heptyl-β-D-, n-octyl-α-D-,n-octyl-β-D-, n-nonyl-β-D-, n-decyl-α-D- andn-decyl-β-D-glucopyranoside. Such glucopyranosides are sometimes termedglucosides. The two non-ionic detergents may be employed in a weightratio from about 1:10 to about 10:1. The use of the two detergentsenables a high degree of recovery of outer membrane antigen which remainsoluble at a wide range of temperature of storage. Alternatively, thetwo detergents may be replaced by sodium dodecyl sulphate.

The procedure described herein for the preparation of the outer membraneextract produces a novel Chlamydial antigen preparation. Accordingly, inan additional aspect of the present invention, there is provided apurified outer membrane antigen extract of a strain of Chlamydia in theform of an aqueous solution of antigen in the presence of at least twonon-ionic detergents and a reducing agent.

In such composition, the purified outer membrane antigen extractcomprises the major outer membrane protein (MOMP) of the strain ofChlamydia, particularly substantially in its native conformation. TheMOMP usually comprises homooligomers and heterooligomers thereof, whichmay have the molecular weights from about 45 to about 125 kDa.

The provision of such novel purified materials enables there to beprovided, in accordance with an additional aspect of the invention, avaccine composition effective for protection against disease caused by astrain of Chlamydia, including Chlamydia trachomatis, comprisingpurified and non-denatured major outer membrane protein (MOMP) of thestrain of Chlamydia substantially in its native conformation.

Such MOMP may be in the form of unaggregated homooligomers andheterooligomers. The vaccine composition may be in the form ofimmunostimulatory complexes (ISCOMs) incorporating the MOMP. The vaccinecomposition may further comprise at least one other chlamydial ornon-chlamydial antigen.

The present invention further extends, in a further aspect of theinvention, to a method for producing a vaccine against disease caused bya strain of Chlamydia, including Chlamydia trachomatis, comprising:

administering the vaccine composition provided herein to a test host todetermine an amount and a frequency of administration thereof to conferprotection against disease caused by the strain of Chlamydia; and

formulating the vaccine in a form suitable for administration to atreated host, which may be a human, in accordance with the determinedamount and frequency of administration.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdescription with reference to the accompanying drawings, in which:

FIG. 1 shows the procedure for the purification of the chlamydialmembrane antigen extract (MAE), in accordance with one aspect of theinvention;

FIGS. 2A-2C, comprising three panels, are SDS-PAGE gels showing theresults of preparing the MAE using octyl glucoside (upper panel), Mega10 (middle panel), or a mixture of the two (lower panel) to extract theouter membrane complexes in the purification scheme of FIG. 1.MW=molecular weight markers (kDa); EB=elementary bodies; Lane 1=solublefraction obtained after Sarkosyl extraction; Lane 2=membrane antigenextract; Lane 3=insoluble fraction containing residual membraneassociated material;

FIG. 3 shows the results of an immunoblot demonstrating the presence ofHSP60 in the Sarkosyl-soluble fraction (lane 1) but not in the MAE (lane3). FIG. 3 also shows the presence in the MAE of MOMP (lanes 2 and 5)and several other antigens (lanes 2 and 4);

FIG. 4 shows the results of an immunoblot conducted under reducing andnon-reducing conditions demonstrating that MOMP in the MAE is present asoligomers (lane NR) with a molecular weights greater than that ofmonomeric MOMP (lane R). The indicated molecular weights (kDa) areactual molecular weights for the materials;

FIGS. 5A-5B, comprising panels A and B, are photomicrographs of ISCOMsprepared using the methods of Morein (ref. 36) (A) or of Mowat and Reid(ref. 37) (B), as described in the Examples below, and provided inaccordance with an aspect of the present invention;

FIGS. 6A-6F, contain bar graphs showing the immunogenicity of ISCOMscontaining MAE administered to mice intra-nasally or intra-peritoneally,in comparison to Freund's adjuvanted MAE administeredintra-peritoneally. ISCOMs administered by either route elicit both IgGand IgA in vaginal secretions;

FIG. 7 contains graphical representations of the response of uninfectedC3 H mice to MAE-ISCOMs for different manners of preparation of theMAE-ISCOMs and different routes of inoculation. Log₁₀ serum IgGanti-MOMP titres are shown at different stages of inoculation; and

FIG. 8 contains graphical representations of the response of previouslyinfected C3 H mice to MAE-ISCOMs for different manners of preparation ofthe MAE-ISCOMs and different routes of inoculation. Log₁₀ serum IgGanti-MOMP titres are shown at different stages of inoculation.

GENERAL DESCRIPTION OF INVENTION

The present invention provides novel techniques which can be employedfor preparing outer membrane antigen extracts from Chlamydia, includingpurified major outer membrane protein from Chlamydia. Any Chlamydiastrain, including C. trachomatis, may be conveniently used to providethe outer membrane antigen extracts as provided herein. Such strains aregenerally available from clinical sources and from bacterial culturecollections.

Referring to FIG. 1, there is illustrated, in the form of a flow chart,a procedure for the purification of the chlamydial outer membraneantigen extract (MAE). Thus, purified elementary bodies (EBs) areresuspended in 10 mM phosphate buffer, pH 7.4, and made to 1 wt %Sarkosyl, 10 mM dithiothreitol (DTT). The mixture is incubated at 37° C.for from about 30 minutes to about 24 hours, with occasional 20-secondpulses in a sonicating water bath. Following the incubation, soluble andinsoluble fractions are separated by centrifugation at 150,000 g for 1hour at 20° C. The insoluble fraction comprises outer membranecomplexes, which are recovered as a pellet, while soluble materialremains in the supernatant. The insoluble fraction is resuspended in 10mM phosphate buffer, pH 7.4, containing 10 mM DTT, anddecanoyl-N-methylglucamide (Mega 10) and/or octyl glucoside at a totalcombined concentration of about 1 wt %. The resuspended material isincubated at 37° C. for from about 30 minutes to about 24 hours, withoccasional 20-second pulses in a sonicating water bath. Following theincubation, soluble and insoluble fractions are separated bycentrifugation at 150,000 g for 1 hour at 20° C. The material remainingin the supernatant is the MAE.

FIG. 2 illustrates the preparation of MAE using octyl glucoside, Mega10, or a mixture of the two detergents, to extract the membrane antigensfrom the Sarkosyl-insoluble pellet. When the MAE is prepared using Mega10 alone, or a mixture of octyl glucoside and Mega 10 in, for example,the ratio 1:3, the final insoluble pellet contains less MOMP than whenonly octyl glucoside is used. MW=molecular weight markers; EB=elementarybodies; Lane 1=soluble fraction obtained after Sarkosyl extraction; Lane2=membrane antigen extract; Lane 3=insoluble fraction containingresidual membrane associated material.

FIG. 3 illustrates the composition of the MAE as determined byimmunoblotting. Using a rabbit antiserum specific for the cytoplasmicprotein HSP60 shows that there is HSP60 in the Sarkosyl-soluble fraction(lane 1) but not in the MAE (lane 3). The major component of the MAE isthe chlamydial major outer membrane protein (MOMP) as shown byimmunoblotting with pooled strain-specific convalescent mouse antisera(lane 2) or with a MOMP-specific monoclonal antibody (lane 5). However,several other antigenic components of the MAE can be demonstrated usingsera raised to the homologous strain of chlamydia (lane 2) or to aheterologous strain (lane 5).

FIG. 4 shows the results of an immunoblot conducted under reducing andnon-reducing conditions. These results demonstrate that MOMP in the MAEis present as oligomers (lane NR) with molecular weights ranging from 45to 125 kDa. Monomeric MOMP of molecular weight about 39,000 Da is shownin Lane R.

FIG. 5 illustrates ISCOMs containing MAE prepared using the methods ofMorein (ref. 36) (A) or of Mowat and Reid (ref. 37) (B). When followingthe method of Morein, ISCOMs are prepared by diluting the MAE to about0.2 mg/mL with l0 mM phosphate buffer pH 6.8. Phosphatidyl choline andcholesterol are dissolved at about 5 mg/mL each in approximately 20%Mega 10 then added to the diluted MAE to a final concentration of about0.2 mg/mL each. Quil A is added to a concentration of about 1 mg/mL.Sufficient 20% Mega 10 is then added to bring the final concentration inthe mixture to about 1%. The mixture is incubated with shaking at roomtemperature overnight then dialysed at 20° to 25° C. against threechanges of 10 mM phosphate buffer, pH 6.8, for from about 2 hours toabout 20 hours per change. When prepared according to this method theISCOMs are uniform particles about 40 to 50 nm in diameter.

When following the method of Mowat and Reid, the membrane antigenextract is adjusted to a protein concentration of about 0.5 to 1 mg/mLand to a detergent concentration of about 2%. Quil A is added to aconcentration of about 1 mg/mL. Phosphatidyl choline and cholesterol aredissolved at about 10 mg each per mL in approximately 2% Mega 10 oroctyl glucoside, then added to the membrane antigen extract at aconcentration of about 0.5 mg each per mL. The mixture is mixed, thendialysed at 20° to 25° C. against six changes of 50 mM Tris-HCl, pH 8.5for about from about 6 hours to about 18 hours per change. When preparedaccording to this method the ISCOMs vary in diameter from about 30 nm toabout 200 nm.

FIG. 6 shows the immunogenicity of ISCOMs containing MAE administered tomice either intranasally or intraperitoneally. Female A/J mice wereinoculated with MAE-ISCOMs containing about 0.25 μg of protein by theintraperitoneal (mice #63-64) or the intranasal (mice #65-68) routes ondays 1 and 14, or with MAE containing about 1 μg of protein in completeFreund's adjuvant on day 1 and with MAE containing about 1 μg of proteinin incomplete Freund's adjuvant on day 14 (mice #9-12). Sera and vaginalwashes were taken on days 0 and 28, and assayed in an ELISA assay forMAE-specific serum IgG, serum IgA, vaginal IgG and vaginal IgA. TheMAE-ISCOMs induce serum IgG titres comparable to those induced by thehigher dose of MAE in Freund's, and consistently induce vaginal IgG andIgA, which MAE in Freund's did not.

FIGS. 7 and 8 show the serum IgG anti-MOMP ELISA antibody responsesobtained from innoculation of uninfected mice (FIG. 7) and micepreviously infected with Chlamydia trachomatis by intranasal andintramuscular innoculation using various MAE-ISCOM preparations. Theimmunization induced specific serum IgG responses in most uninfectedanimals immmunized with a vaccine containing MAE. Previously-infectedanimals had high pre-existing specific serum IgG responses whichincreased modestly following intramuscular immunization.

In the experiments performed, the intra-muscular route was moreeffective than the intranasal route at inducing serum IgG responses.While serum specific IgG responses were observed using the MAE-ISCOMsprovided herein, animals immunized with ISCOM matrix alone did notproduce a specific serum IgG response.

Histological studies were also carried out and inflammatory lesions inthe uterus and oviducts of immunized mice were assessed as absent(normal tissue), mild or severe, according to the criteria outlined inTable 1 below. The results obtained are set forth in Table 2 below. Asdetailed below, certain groups of mice which had received MAE-ISCOMswere significantly protected from the development of lesions.

Advantages of the MAE-ISCOMs of the present invention include thecapability to induce a strong and protective anti-chlamydial immuneresponse when administered to a mammal without exacerbating chlamydialdisease, by, for example, potentiation of chlamydial disease bysensitising vaccinees to HSP60.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of Chlamydia infections andthe generation of immunological reagents. A further non-limitingdiscussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from immunogenic outer membrane antigen extracts of Chlamydia,including the major outer membrane protein (MOMP), as disclosed herein.The immunogenic composition elicits an immune response which producesantibodies, including anti-MOMP antibodies, IgG and IgA antibodies, andantibodies that are opsonizing or bactericidal. Should the vaccinatedsubject be challenged by Chlamydia, the antibodies bind thereto andthereby inactivate the Chlamydia. Furthermore, opsonizing orbactericidal antibodies may also provide protection by alternativemechanisms. The immunogenic compositions also produce cell mediatedimmune responses including CD4+ and CD8+ T cell response includingcytotoxic T cell responses specific for MOMP.

Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The outer membraneantigen extracts may be mixed with pharmaceutically acceptableexcipients which are compatible therewith. Such excipients may include,water, saline, dextrose, glycerol, ethanol, and combinations thereof.The immunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof. Immunogeniccompositions and vaccines may be administered parenterally, by injectionsubcutaneously or intramuscularly. Alternatively, the immunogeniccompositions formed according to the present invention, may beformulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Such suppositories may be formed from mixtures containingthe active ingredient(s) in the range of about 0.5 to about 10%,preferably about 1 to 2%. Oral formulations may include normallyemployed incipients such as, for example, pharmaceutical grades ofsaccharine, cellulose and magnesium carbonate. These compositions cantake the form of solutions, suspensions, tablets, pills, capsules,sustained release formulations or powders and contain about 1 to 95% ofthe outer membrane antigen extract, preferably about 20 to about 75%.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizeantibodies and if needed, to produce a cell-mediated immune response.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges arereadily determinable by one skilled in the art and may be of the orderof micrograms of the outer membrane antigen extract per vaccination.Suitable regimes for initial administration and booster doses are alsovariable, but may include an initial administration followed bysubsequent administrations. The dosage may also depend on the route ofadministration and will vary according to the size of the host.

The concentration of the outer membrane antigen extracts in animmunogenic composition according to the invention is in general about 1to 95%. A vaccine which contains antigenic material of only one pathogenis a monovalent vaccine. Vaccines which contain antigenic material ofseveral pathogens are combined vaccines and also belong to the presentinvention. Such combined vaccines contain, for example, material fromvarious pathogens or from various strains of the same pathogen, or fromcombinations of various pathogens.

Thus, the immunogenic compositions provided herein may be formulated tocomprise at least one additional immunogen, which may comprise or bederived from bacterial, viral or protozal pathogens including aparamyxovirus, polio, hepatitis B, bacterial toxoids, includingdiphtheria toxoid and tetanus toxoid, influenza, haemophilus, pertussis,pneumococcus, mycobacteria, hepatitis A, HIV, HSV, Neisseria gonorrhoea,Treponema pallidum, and organisms which result in other sexuallytransmitted diseases.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune responses. Thus,adjuvants have been identified that enhance the immune response toantigens delivered parenterally. Some of these adjuvants are toxic,however, and can cause undesirable side-effects, making them unsuitablefor use in humans and many animals. Indeed, only aluminum hydroxide andaluminum phosphate (collectively commonly referred to as alum) areroutinely used as adjuvants in human and veterinary vaccines. Theefficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established and a HBsAg vaccine has beenadjuvanted with alum. While the usefulness of alum is well establishedfor some applications, it has limitations. For example, alum isineffective for influenza vaccination and inconsistently elicits a cellmediated immune response. The antibodies elicited by alum-adjuvantedantigens are mainly of the IgG1 isotype in the mouse, which may not beoptimal for protection by some vaccinal agents.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's complete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

2. Immunoassays

The outer membrane antigen extracts of the present invention are usefulas immunogens for the generation of anti-Chlamydia antibodies and asantigens in immunoassays including enzyme-linked immunosorbent assays(ELISA), RIAs and other non-enzyme linked antibody binding assays orprocedures known in the art for the detection of anti-Chlamydiaantibodies. In ELISA assays, the outer membrane antigen extract isimmobilized onto a selected surface, for example, a surface capable ofbinding proteins such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed antigen, a nonspecificprotein such as a solution of bovine serum albumin (BSA) that is knownto be antigenically neutral with regard to the test sample may be boundto the selected surface. This allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from 2 to 4 hours, at temperatures suchas of the order of about 25° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween or a borate buffer. Following formation of specificimmunocomplexes between the test sample and the bound outer membraneantigen extract, and subsequent washing, the occurrence, and evenamount, of immunocomplex formation may be determined by subjecting theimmunocomplex to a second antibody having specificity for the firstantibody. If the test sample is of human origin, the second antibody isan antibody having specificity for human immunoglobulins and in generalIgG. To provide detecting means, the second antibody may have anassociated activity such as an enzymatic activity that will generate,for example, a colour development upon incubating with an appropriatechromogenic substrate. Quantification may then be achieved by measuringthe degree of colour generation using, for example, a spectrophotometer.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of protein biochemistry and immunology used but not explicitlydescribed in this disclosure and these Examples are amply reported inthe scientific literature and are well within the ability of thoseskilled in the art.

EXAMPLE 1

This Example illustrates the preparation of the membrane antigen extractfrom chlamydial elementary bodies, as shown in FIG. 1.

Purified elementary bodies (EBs), prepared as described in ref. 19, wereresuspended in 10 mM phosphate buffer, pH 7.4, and made to 1 wt %Sarkosyl (N-Lauylsarosine, sodium salt), 10 mM DTT. The EBs wereincubated at 37° C. for about 90 minutes, with occasional 20 secondpulses in a sonicating water bath. Following the incubation, soluble andinsoluble fractions were separated by centrifugation at 150,000 g for 1hour at 20° C. The insoluble fraction comprises outer membrane complexeswhich are recovered as a pellet, while soluble material comprisingprincipally cytoplasmic proteins including HSP60, remains in thesupernatant. The insoluble fraction was resuspended in 10 mM phosphatebuffer, pH 7.4, containing 10 mM DTT, and decanoyl-N-methylglucamide(Mega 10) and/or octyl glucoside at a total combined concentration ofabout 1 wt %. The resuspended material was incubated at 37° C. for about90 minutes, with occasional 20-second pulses in a sonicating water bath.Following the incubation, soluble and insoluble fractions were separatedby centrifugation at 150,000 g for 1 hour at 20° C. The soluble materialremaining in the supernatant was the membrane antigen extract while theinsoluble fraction contained residual membrane-associated material.

EXAMPLE 2

This Example illustrates the preparation of ISCOMs with the membraneantigen extract according to the methods of Morein (ref. 36) or of Mowatand Reid (ref. 37).

When following the method of Morein, ISCOMs were prepared by dilutingthe MAE, prepared as described in Example 1, to about 0.2 mg/ml with 10mM phosphate buffer pH 6.8. Phosphatidyl choline and cholesterol weredissolved at about 5 mg/ml each in approximately 20% Mega 10 and thenadded to the diluted MAE to a final concentration of 0.2 mg/ml each.Quil A (a complex but purified mixture of Quillaja saponins which areglycosides of quillaic acid and carbohydrates) was added to aconcentration of about 1 mg/ml. Sufficient 20% Mega 10 was then added tobring the final concentration in the mixture to about 1%wt. The mixturewas shaken at room temperature overnight and then dialysed at 20 to 25°C. against three changes of 10 mM phosphate buffer, pH 6.8, for about 6hours, about 16 hours and about 6 hours for the three buffer changes.When prepared according to this method, the ISCOMs were uniformparticles about 40 to 50 nm in diameter.

When following the method of Mowat and Reid, the membrane antigenextract, prepared as described in Example 1, was diluted to a proteinconcentration of about 0.5 to 1 mg/ml and to a detergent concentrationof about 2 wt %. Quil was added to a concentration of about 1 mg/ml.Phosphatidyl choline and cholesterol were dissolved at about 10 mg eachper ml in approximately 2% Mega 10 or octyl glucoside, then added to themembrane antigen extract at a concentration of about 0.5 mg each per ml.The mixture was mixed briefly, then dialysed at 20 to 25° C. against sixchanges of 50 mM Tris-HCl, pH 8.5 alternately for about 6 hours andabout 18 hours per buffer change. When prepared according to thismethod, the ISCOMs vary in diameter from about 30 nm to about 200 nm.

Electron micrographs of ISCOMs formed by both methods are shown in FIG.5. Panel A shows the ISCOMs prepared according to the procedure ofMorein and Panel B shows the ISCOMs prepared according to the procedureof Mowat and Reid.

EXAMPLE 3

This Example illustrates the immunogenicity of chlamydial membraneantigen extract (MAE)-ISCOMs in mice.

Female A/J mice were immunized with MAE-ISCOMs, prepared as described inExample 2 following the procedure of Mowat and Reid, containing about0.25 μg of protein by the intraperitoneal (mice #63-64) or theintranasal (mice #65-68) routes on days 1 and 14, or with MAE containingabout 1 μg of protein in complete Freund's adjuvant on day 1 and withMAE containing about 1 μg of protein in incomplete Freund's adjuvant onday 14 (mice #9-12). Sera and vaginal washes were taken on days 0 and28, and assayed in an ELISA assay for MAE-specific serum IgG, serum IgA,vaginal IgG and vaginal IgA.

As may be seen from the results obtained (FIG. 6), the MAE-ISCOMsprovided herein induced serum IgG titres comparable to those induced bythe higher dose of MAE in Freund's (upper panels), and consistentlyinduced vaginal IgG (penultimate panels). MAE in Freund's adjuvant didnot induce any IgA antibodies whereas the MAE-ISCOMs produced IgAantibodies (lower panels).

EXAMPLE 4

This Example illustrates the use of MAE-ISCOMs to protect mice fromchlamydial infection.

A group of 160 female mice C3 H, aged 6 to 8 weeks, were divided intotwo groups of 80, designated infected and uninfected. On days 0 and 7,all mice were treated with 2.5 mg progesterone administeredsubcutaneously. On day 7, the infected group was vaginally inoculatedwith 1000 ID₅₀ of Chlamydia trachomatis MoPn strain and the uninfectedgroup was vaginally inoculated with SPG buffer. Inoculations wereperformed under light anaesthesia. The animals were then rested untilday 91, when they were further divided into sixteen groups andvaccinated as follows:

10 infected mice received intramuscularly MAE-ISCOMs prepared by theprocedure of Morein as described in Example 2,

10 infected mice received intranasally MAE-ISCOMs prepared by theprocedure of Morein as described in Example 2,

10 infected mice received MAE-ISCOMs, prepared by the procedure ofMorein as described in Example 2, in which the MAE was prepared asdescribed in Example 1 except that sodium dodecyl sulphate was used inplace of Mega 10 and/or octyl glucoside, intramuscularly,

10 infected mice received MAE-ISCOMs, prepared by the procedure ofMorein as described in Example 2, in which the MAE was prepared asdescribed in Example 1 except that sodium dodecyl sulphate was used inplace of Mega 10 and/or octyl glucoside, intranasally,

10 infected mice received MAE, prepared as described in Example 1, mixedwith ISCOM matrix, intramuscularly,

10 infected mice received MAE, prepared as described in Example 1, mixedwith ISCOM matrix, intranasally,

10 infected mice received ISCOM matrix intramuscularly,

10 infected mice received ISCOM matrix intranasally,

10 uninfected mice received intramuscularly MAE-ISCOMs, prepared by theprocedure of Morein as described in Example 2,

10 uninfected mice received intranasally MAE-ISCOMs, prepared by theprocedure of Morein as described in Example 2,

10 uninfected mice received MAE-ISCOMs, prepared by the procedure ofMorein as described in Example 2, in which the MAE was prepared asdescribed in Example 1 except that sodium dodecyl sulphate was used inplace of Mega 10 and/or octyl glucoside, intramuscularly,

10 uninfected mice received MAE-ISCOMs, prepared by the procedure ofMorein as described in Example 2, in which the MAE was prepared asdescribed in Example 1 except that sodium dodecyl sulphate was used inplace of Mega 10 and/or octyl glucoside, intranasally,

10 uninfected mice received MAE, prepared as described in Example 1,mixed with ISCOM matrix, intramuscularly,

10 uninfected mice received MAE, prepared as described in Example 1,mixed with ISCOM matrix, intranasally,

10 uninfected mice received ISCOM matrix intramuscularly,

10 uninfected mice received ISCOM matrix intranasally.

The ISCOM matrix employed was prepared in the same way as the MAE-ISCOMsas in Example 2 except that MAE was omitted from the reaction mixture.MAE mixed with ISCOM matrix was prepared by adding MAE to preformedISCOM matrix. Each dose of vaccine contained about 2 μg of MAE and about10 μg of saponin. These vaccinations were repeated on about days 112 and133. Blood and vaginal washes were taken just before each vaccinationand assayed in an ELISA assay for antigen specific serum IgG. As may beseen from the results obtained (FIGS. 7, 8) the immunizations inducedspecific serum IgG responses in most uninfected animals immunized with avaccine containing MAE. Previously infected animals had highpre-existing specific serum IgG responses as a consequence of theinfection which increased modestly following intramuscullarimmunization. The intra-muscular route was more affective than theintranasal route at inducing specific serum IgG responses. MAE-ISCOMsprepared by the procedure of Morein as described in Example 2 were moreeffective at inducing specific serum IgG responses than MAE-ISCOMs inwhich the MAE was prepared as described in Example 1 except that sodiumdodecyl sulphate was used in place of Mega 10 and/or octyl glucoside.Animals immunized with ISCOM matrix alone did not produce a specificserum IgG response.

All mice were challenged with about 100 ID₅₀ of Chlamydia trachomatisMoPn strain, administered vaginally to anaesthetized animals on aboutdays 145, 147 and 149. On about days 154 and 161 three mice from each ofthe 12 groups were necropsied. On day 168 all remaining mice werenecropsied. At necropsy, the reproductive tract was removed and dividedinto parts so that symptoms due to infection were determined byexamination of histological sections.

Inflammatory lesions in the uterus and oviducts were assessed as absent(normal tissue), mild or severe, according to the criteria in Table 1.As may be seen from the results shown in Table 2, the following groupsof mice were significantly protected from the development of lesions:

uninfected mice which received MAE-ISCOMs, prepared by the procedure ofMorein as described in Example 2, intramuscularly;

infected mice which received MAE-ISCOMs, prepared by the procedure ofMorein as described in Example 2, intramuscularly;

infected mice which received MAE-ISCOMs, prepared by the procedure ofMorein as described in Example 2, intranasally;

infected mice which received MAE-ISCOMs, in which the MAE was preparedas described in example 1 except that sodium dodecyl sulphate was usedin place of Mega 10 and/or octyl glucoside, intramuscularly; and

infected mice which received MAE prepared as described in Example 1mixed with ISCOM matrix, intramuscularly.

The presence of chlamydiae in the tract can be assessed immunologicallyand by PCR assay.

SUMMARY OF INVENTION

In summary of this disclosure, the present invention provides ISCOM,chlamydial major outer membrane protein complexes, useful in vaccinesagainst chlamydial diseases, and in the preparation of immunologicalreagents. Modifications are possible within the scope of the invention.

TABLE 1 LESION SEVERITY LESION DESCRIPTION UTERUS Absent Normal uterus.(May have occasional mild foci of inflammation) Mild Mild to moderateinflammation of the tissues but little or no infiltrate in the uterinelumen. Severe Moderate to severe tissue inflammation with widespreadinfiltration into the uterine lumen. OVIDUCTS Absent Normal oviduct.Mild Mild inflammation of oviduct walls and supporting tissues, may havea few leukocytes in the oviduct lumen. Tissue architecture essentiallynormal. OR Moderate and widespread inflammation of oviduct walls andsupporting tissues. Usually some localised infiltration into the oviductlumen, but little damage to lumenal epithelium. Severe Extensiveinfiltration into the oviduct lumen. Lumenal epithelium still present,but microvilli flattened or absent. OR Extensive infiltration into theoviduct lumen. Lumenal epithelium absent or severely damaged.

TABLE 2 ROUTE OF IMMUNIZATION INTRANASAL INTRAMUSCULAR Lesions LesionsADJUVANT ANTIGEN Absent Mild Severe Absent Mild Severe (1) UninfectedC3H mice Formulated MAE 4/9  5/9  0/9  8/9 0/9 1/9 ISCOMs *0.024Formulated MAE prepared 5/10 2/10 3/10 5/8 3/8 0/8 ISCOMs using SDSISCOM Matrix MAE 5/10 4/10 1/10  3/10  6/10  1/10 ISCOM Matrix None 3/8 0/8  5/8  4/9 4/9 1/9 2) Previously infected C3H mice Formulated MAE5/10 5/10 0/10 3/7 4/7 0/7 ISCOMs *0.009 *0.042 Formulated MAE prepared0/10 8/10 2/10  4/10  6/10  0/10 ISCOMS using SDS *0.031 ISCOM MatrixMAE 1/8  7/8  0/8  5/9 4/9 0/9 *0.005 ISCOM Matrix None 1/10 9/10 0/10 0/10 10/10  0/10 * = p values versus combined controls

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What we claim is:
 1. A method of producing an outer membrane antigenextract of a strain of Chlamydia, which comprises: detergent extractingelementary bodies of said strain of Chlamydia in the presence of areducing agent to solubilize cytoplasmic material away from outermembrane material; separating said solubilized cytoplasmic material fromthe outer membrane materials; detergent extracting said outer membranematerial using at least two non-ionic detergents in the presence of areducing agent to solubilize outer membrane antigens; and separatingsaid solubilized outer membrane antigens from residual unextractedmembrane-associated material to provide said outer membrane antigenextract.
 2. The method of claim 1 wherein said at least two non-ionicdetergents comprise a N-methylglucamide non-ionic detergent and aglucopyranoside non-ionic detergent.
 3. The method of claim 2 whereinsaid N-methylglucamide non-ionic detergent is selected from the groupconsisting of heptanoyl-, octanoyl-, nonanoyl- anddecanoyl-N-methylglucamide.
 4. The method of claim 3 wherein saidglucopyranoside non-ionic detergent is selected from the groupconsisting of n-hexyl-β-D, n-heptyl-β-D, n-octyl-α-D-, n-octyl-β-D,n-nonyl-β-D, n-decyl-α-D- and n-decyl-α-D-glucopyranoside.
 5. The methodof claim 4 wherein said two non-ionic detergents are employed in aweight-ratio from about 1:10 to about 10:1.